IL-10 regulates murine lupus

MRL/MpJ-Tnfrsf6(lpr) (MRL/MpJ-Fas(lpr); MRL-Fas(lpr)) mice develop a spontaneous lupus syndrome closely resembling human systemic lupus erythematosus. To define the role of IL-10 in the regulation of murine lupus, IL-10 gene-deficient (IL-10(-/-)) MRL-Fas(lpr) (MRL-Fas(lpr) IL-10(-/-)) mice were generated and their disease phenotype was compared with littermates with one or two copies of an intact IL-10 locus (MRL-Fas(lpr) IL-10(+/-) and MRL-Fas(lpr) IL-10(+/+) mice, respectively). MRL-Fas(lpr) IL-10(-/-) mice developed severe lupus, with earlier appearance of skin lesions, increased lymphadenopathy, more severe glomerulonephritis, and higher mortality than their IL-10-intact littermate controls. The increased severity of lupus in MRL-Fas(lpr) IL-10(-/-) mice was closely associated with enhanced IFN-gamma production by both CD4(+) and CD8(+) cells and increased serum concentration of IgG2a anti-dsDNA autoantibodies. The protective effect of IL-10 in this lupus model was further supported by the observation that administration of rIL-10 reduced IgG2a anti-dsDNA autoantibody production in wild-type MRL-Fas(lpr) animals. In summary, our results provide evidence that IL-10 can down-modulate murine lupus through inhibition of pathogenic Th1 cytokine responses. Modulation of the level of IL-10 may be of potential therapeutic benefit for human lupus.     

PD-1 protects against inflammation and myocyte damage in T cell-mediated myocarditis

PD-1, a member of the CD28 family of immune regulatory molecules, is expressed on activated T cells, interacts with its ligands, PD-L1/B7-H1 and PD-L2/B7-DC, on other cells, and delivers inhibitory signals to the T cell. We studied the role of this pathway in modulating autoreactive T cell responses in two models of myocarditis. In a CD8(+) T cell-mediated adoptive transfer model, we found that compared with Pd1(+/+) CD8(+) T cells, Pd1(-/-) CD8(+) T cells cause enhanced disease, with increased inflammatory infiltrate, particularly rich in neutrophils. Additionally, we show enhanced proliferation in vivo and enhanced cytotoxic activity of PD-1-deficient T lymphocytes against myocardial endothelial cells in vitro. In experimental autoimmune myocarditis, a disease model dependent on CD4(+) T cells, we show that mice lacking PD-1 develop enhanced disease compared with wild-type mice. PD-1-deficient mice displayed increased inflammation, enhanced serum markers of myocardial damage, and an increased infiltration of inflammatory cells, including CD8(+) T cells. Together, these studies show that PD-1 plays an important role in limiting T cell responses in the heart.     

Gammadelta T cells promote a Th1 response during coxsackievirus B3 infection in vivo: role of Fas and Fas ligand

Fas/Fas ligand (FasL) interactions regulate disease outcome in coxsackievirus B3 (CVB3)-induced myocarditis. MRL(+/+) mice infected with CVB3 develop severe myocarditis, a dominant CD4(+) Th1 (gamma interferon [IFN-gamma(+)]) response to the virus, and a predominance of gammadelta T cells in the myocardial infiltrates. MRL lpr/lpr and MRL gld/gld mice, which lack normal expression of Fas and express a mutated FasL, respectively, have minimal myocarditis and show a dominant CD4(+) Th2 (interleukin-4 [IL-4(+)]) phenotype to CVB3. Spleen cells from virus-infected wild-type, lpr, and gld animals proliferate equally to virus in vitro. Adoptive transfer of gammadelta T cells from hearts of CVB3-infected MRL(+/+) mice (FasL(+)) into infected MRL gld/gld recipients (FasL(-)/Fas(+)) restores both disease susceptibility and Th1 cell phenotype. However, transfer of these cells into MRL lpr/lpr recipients (FasL(+)/Fas(-)) did not promote myocarditis and the viral response remained Th2 biased. This paralleled the expression of very high surface levels of FasL by myocardial gammadelta T cells, as well as their propensity to selectively lyse Th2 virus-specific CD4(+) T cells. These results demonstrate that Fas/FasL interactions conferred by gammadelta T cells on lymphocyte subpopulations may regulate the cytokine response to CVB3 infection and pathogenicity.     

Dicer insufficiency and microRNA-155 overexpression in lupus regulatory T cells: an apparent paradox in the setting of an inflammatory milieu

Systemic lupus erythematosus is a chronic autoimmune disease characterized by loss of tolerance to self-Ags and activation of autoreactive T cells. Regulatory T (Treg) cells play a critical role in controlling the activation of autoreactive T cells. In this study, we investigated mechanisms of potential Treg cell defects in systemic lupus erythematosus using MRL-Fas(lpr/lpr) (MRL/lpr) and MRL-Fas(+/+) mouse models. We found a significant increase in CD4(+)CD25(+)Foxp3(+) Treg cells, albeit with an altered phenotype (CD62L(-)CD69(+)) and with a reduced suppressive capacity, in the lymphoid organs of MRL strains compared with non-autoimmune C3H/HeOuj mice. A search for mechanisms underlying the altered Treg cell phenotype in MRL/lpr mice led us to find a profound reduction in Dicer expression and an altered microRNA (miRNA, miR) profile in MRL/lpr Treg cells. Despite having a reduced level of Dicer, MRL/lpr Treg cells exhibited a significant overexpression of several miRNAs, including let-7a, let-7f, miR-16, miR-23a, miR-23b, miR-27a, and miR-155. Using computational approaches, we identified one of the upregulated miRNAs, miR-155, that can target CD62L and may thus confer the altered Treg cell phenotype in MRL/lpr mice. In fact, the induced overexpression of miR-155 in otherwise normal (C3H/HeOuj) Treg cells reduced their CD62L expression, which mimics the altered Treg cell phenotype in MRL/lpr mice. These data suggest a role of Dicer and miR-155 in regulating Treg cell phenotype. Furthermore, simultaneous appearance of Dicer insufficiency and miR-155 overexpression in diseased mice suggests a Dicer-independent alternative mechanism of miRNA regulation under inflammatory conditions.     

Macrophage migration inhibitory factor deficiency attenuates macrophage recruitment, glomerulonephritis, and lethality in MRL/lpr mice

Systemic lupus erythematosus (SLE) is a serious systemic autoimmune disease of unknown etiology. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that is operative in innate and adaptive immunity and important in immune-mediated diseases such as rheumatoid arthritis and atherosclerosis. The functional relevance of MIF in systemic autoimmune diseases such as SLE is unknown. Using the lupus-prone MRL/lpr mice, we aim to examine the expression and function of MIF in this murine model of systemic autoimmune disease. These experiments revealed that renal MIF expression was significantly higher in MRL/lpr mice compared with nondiseased control mice (MRL/MpJ), and MIF was also markedly up-regulated in skin lesions of MRL/lpr mice. To examine the effect of MIF on development of systemic autoimmune disease, we generated MRL/lpr mice with a targeted disruption of the MIF gene (MIF(-/-)MRL/lpr), and compared their disease manifestations to MIF(+/+)MRL/lpr littermates. MIF(-/-)MRL/lpr mice exhibited significantly prolonged survival, and reduced renal and skin manifestations of SLE. These effects occurred in the absence of major changes in T and B cell markers or alterations in autoantibody production. In contrast, renal macrophage recruitment and glomerular injury were significantly reduced in MIF(-/-)MRL/lpr mice, and this was associated with reduction in the monocyte chemokine MCP-1. Taken together, these data suggest MIF as a critical effector of organ injury in SLE.     

C1q deficiency and autoimmunity: the effects of genetic background on disease expression

Gene-targeted C1q-deficient mice have been shown to develop a syndrome reminiscent of human systemic lupus erythematosus with antinuclear Abs and proliferative glomerulonephritis. Initial phenotypic analysis conducted in (129 x C57BL/6) hybrid mice showed that background genes were a significant factor for the full expression of the autoimmune disease. To assess the contribution of background genes in the expression of the autoimmune phenotype, the disrupted C1qa gene was backcrossed for seven generations onto C57BL/6 and MRL/Mp(+/+) strains. These were intercrossed with C57BL/6.lpr/lpr and MRL/Mp-lpr/lpr strains to generate C1q-deficient substrains. In C1q-deficient C57BL/6 mice, no evidence of an autoimmune phenotype was found, and C1q deficiency in both the C57BL/6.lpr/lpr and MRL/Mp-lpr/lpr strains did not modify the autoimmune phenotype observed in wild-type controls. However, in C1q-deficient MRL/Mp(+/+) animals an acceleration of both the onset and the severity of antinuclear Abs and glomerulonephritis was seen. Disease was particularly pronounced in females, which developed severe crescentic glomerulonephritis accompanied by heavy proteinuria. In addition, the C1q-deficient MRL/Mp(+/+) mice had an impairment in the phagocytic clearance of apoptotic cells in vivo. These data demonstrate that the expression of autoimmunity in C1q-deficient mice is strongly influenced by other background genes. The work also highlights the potential value of the C1q-deficient MRL/Mp(+/+) strain as a tool with which to dissect further the underlying mechanisms of the autoimmune syndrome associated with C1q deficiency.     

Negative role of colony-stimulating factor-1 in macrophage, T cell, and B cell mediated autoimmune disease in MRL-Fas(lpr) mice

Inflammation in the kidney and other tissues (lung, and salivary and lacrimal glands) is characteristic of MRL-Fas(lpr) mice with features of lupus. Macrophages (Mphi) are prominent in these tissues. Given that 1) Mphi survival, recruitment, proliferation, and activation during inflammation is dependent on CSF-1, 2) Mphi mediate renal resident cell apoptosis, and 3) CSF-1 is up-regulated in MRL-Fas(lpr) mice before, and during nephritis, we hypothesized that CSF-1-deficient MRL-Fas(lpr) mice would be protected from Mphi-mediated nephritis, and the systemic illness. To test this hypothesis, we compared CSF-1-deficient MRL-Fas(lpr) with wild-type strains. Renal pathology is suppressed and function improved in CSF-1-deficient MRL-Fas(lpr) mice. There are far fewer intrarenal Mphi and T cells in CSF-1-deficient MRL-Fas(lpr) vs wild-type kidneys. This leukocytic reduction results from suppressed infiltration, and intrarenal proliferation, but not enhanced apoptosis. The CSF-1-deficient MRL-Fas(lpr) kidneys remain preserved as indicated by greatly reduced indices of injury (nephritogenic cytokines, tubular apoptosis, and proliferation). The renal protective mechanism in CSF-1-deficient mice is not limited to reduced intrarenal leukocytes; circulating Igs and autoantibodies, and renal Ig deposits are decreased. This may result from enhanced B cell apoptosis and fewer B cells in CSF-1-deficient MRL-Fas(lpr) mice. Furthermore, the systemic illness including, skin, lung, and lacrimal and salivary glands pathology, lymphadenopathy, and splenomegaly are dramatically suppressed in CSF-1-deficient MRL-Fas(lpr) as compared with wild-type mice. These results indicate that CSF-1 is an attractive therapeutic target to combat Mphi-, T cell-, and B cell-mediated autoimmune lupus.     

Decreased expression of the Ets family transcription factor Fli-1 markedly prolongs survival and significantly reduces renal disease in MRL/lpr mice

Increased Fli-1 mRNA is present in PBLs from systemic lupus erythematosus patients, and transgenic overexpression of Fli-1 in normal mice leads to a lupus-like disease. We report in this study that MRL/lpr mice, an animal model of systemic lupus erythematosus, have increased splenic expression of Fli-1 protein compared with BALB/c mice. Using mice with targeted gene disruption, we examined the effect of reduced Fli-1 expression on disease development in MRL/lpr mice. Complete knockout of Fli-1 is lethal in utero. Fli-1 protein expression in heterozygous MRL/lpr (Fli-1(+/-)) mice was reduced by 50% compared with wild-type MRL/lpr (Fli-1(+/+)) mice. Fli-1(+/-) MRL/lpr mice had significantly decreased serum levels of total IgG and anti-dsDNA Abs as disease progressed. Fli-1(+/-) MRL/lpr mice had significantly increased splenic CD8(+) and naive T cells compared with Fli-1(+/+) MRL/lpr mice. Both in vivo and in vitro production of MCP-1 were significantly decreased in Fli-1(+/-) MRL/lpr mice. The Fli-1(+/-) mice had markedly decreased proteinuria and significantly lower pathologic renal scores. At 48 wk of age, survival was significantly increased in the Fli-1(+/-) MRL/lpr mice, as 100% of Fli-1(+/-) MRL/lpr mice were alive, in contrast to only 27% of Fli-1(+/+) mice. These findings indicate that Fli-1 expression is important in lupus-like disease development, and that modulation of Fli-1 expression profoundly decreases renal disease and improves survival in MRL/lpr mice.     

Deficiency in beta(2)-microglobulin, but not CD1, accelerates spontaneous lupus skin disease while inhibiting nephritis in MRL-Fas(lpr) nice: an example of disease regulation at the organ level

When mutations that inactivate molecules that function in the immune system have been crossed to murine lupus strains, the result has generally been a uniform up-regulation or down-regulation of autoimmune disease in the end organs. In the current work we report an interesting dissociation of target organ disease in beta(2)-microglobulin (beta(2)m)-deficient MRL-Fas(lpr) (MRL/lpr) mice: lupus skin lesions are accelerated, whereas nephritis is ameliorated. beta(2)m deficiency affects the expression of classical and nonclassical MHC molecules and thus prevents the normal development of CD8- as well as CD1-dependent NK1(+) T cells. To further define the mechanism by which beta(2)m deficiency accelerates skin disease, we studied CD1-deficient MRL/lpr mice. These mice do not have accelerated skin disease, excluding a CD1 or NK1(+) T cell-dependent mechanism of beta(2)m deficiency. The data indicate that the regulation of systemic disease is not solely governed by regulation of initial activation of autoreactive lymphocytes in secondary lymphoid tissue, as this is equally relevant to renal and skin diseases. Rather, regulation of autoimmunity can also occur at the target organ level, explaining the divergence of disease in skin and kidney in beta(2)m-deficient mice.     

Abnormal IgG galactosylation and arthritis in MRL-Fas(lpr) or MRL-FasL(gld) mice are under the control of the MRL genetic background.

MRL mice bearing the lpr (Fas) or gld (Fas ligand) mutation, MRL-Fas(lpr) or MRL-FasL(gld), respectively, develop arthritis similar to rheumatoid arthritis, but C3H and C57BL/6 mice bearing such mutations do not. In MRL-Fas(lpr) mice, agalactosylated oligosaccharides in serum IgG increase significantly in comparison to MRL-+/+ mice without arthritis. In this study, an increased level of agalactosylation in IgG, as compared to MRL-+/+, was found in both MRL-Fas(lpr) and MRL-FasL(gld) mice. In contrast, the incidence of IgG without galactose was comparable among C3H-Fas(lpr), C3H-FasL(gld), and C3H-+/+ mice as well as between C57BL/6-Fas(lpr) and C57BL/6-+/+ mice. These results suggest that the increase in agalactosylated IgG and the development of arthritis in MRL-Fas(lpr) and MRL-FasL(gld) mice are controlled by the MRL genetic background.     

Terminal deoxynucleotidyl transferase deficiency decreases autoimmune disease in MRL-Fas(lpr) mice.

The neonatal Ab and TCR repertoires are much less diverse, and also very different from, the adult repertoires due to the delayed onset of terminal deoxynucleotidyl transferase (TdT) expression in ontogeny. TdT adds nontemplated N nucleotides to the junctions of Igs and TCRs, and thus its absence removes one of the major components of junctional diversity in complementarity-determining region 3 (CDR3). We have generated TdT-deficient MRL/lpr, Fas-deficient (MRL-Fas(lpr)) mice, and show that they have an increased lifespan, decreased incidence of skin lesions, and much lower serum levels of anti-dsDNA, anti-chromatin, and IgM rheumatoid factors. The generalized hypergammaglobulinemia characteristic of MRL-Fas(lpr) mice is also greatly reduced, as is the percentage of CD4(-)CD8(-)B220(+) (double-negative) T cells. IgG deposits in the kidney are significantly reduced, although evidence of renal disease is present in many mice at 6 mo. CDR3 regions of both IgH and TCR from peripheral lymphocytes of MRL-Fas(lpr) mice are shorter in the absence of TdT, and there is a paucity of arginines in the IgH CDR3 regions of the MRL-Fas(lpr) TdT(-/-) mice. Because the amelioration of symptoms is so widespread, it is likely that the absence of N regions has more of an affect than merely decreasing the precursor frequency of anti-dsDNA B cells. Hence, either the T or B cell repertoires, or more likely both, require N region diversity to produce the full spectrum of autoimmune lupus disease.     

Critical role of the alpha 4 integrin/VCAM-1 pathway in cerebral leukocyte trafficking in lupus-prone MRL/fas(lpr) mice

MRL/fas(lpr) mice are affected by a systemic autoimmune disease that results in leukocyte recruitment to a wide range of vascular beds, including the cerebral microvasculature. The mechanisms responsible for the leukocyte trafficking to the brain in these animals are not known. Therefore, the aim of this study was to directly examine the cerebral microvasculature in MRL/fas(lpr) mice and determine the molecular mechanisms responsible for this leukocyte recruitment. Intravital microscopy was used to assess leukocyte-endothelial cell interactions (rolling, adhesion) in the pial microcirculation of MRL(+/+) (control) and MRL/fas(lpr) mice at 8, 12, and 16 wk of age. Leukocyte rolling and adhesion were rarely observed in MRL(+/+) mice of any age. MRL/fas(lpr) mice displayed similar results at 8 and 12 wk. However, at 16 wk, significant increases in leukocyte rolling and adhesion were observed in these mice. Histological analysis revealed that the interacting cells were exclusively mononuclear. Leukocyte rolling was reduced, but not eliminated in P-selectin(-/-)-MRL/fas(lpr) mice. However, leukocyte adhesion was not reduced in these mice, indicating that P-selectin-dependent rolling was not required for leukocyte recruitment to the cerebral vasculature in this model of systemic inflammation. E-selectin blockade also had no effect on leukocyte rolling. In contrast, blockade of either the alpha4 integrin or VCAM-1 eliminated P-selectin-independent leukocyte rolling. alpha4 Integrin blockade also significantly inhibited leukocyte adhesion. These studies demonstrate that the systemic inflammatory response that affects MRL/fas(lpr) mice results in leukocyte rolling and adhesion in the cerebral microcirculation, and that the alpha4 integrin/VCAM-1 pathway plays a central role in mediating these interactions.     

Aberrant macrophages mediate defective kidney repair that triggers nephritis in lupus-susceptible mice

CSF-1, required for macrophage (M?) survival, proliferation, and activation, is upregulated in the tubular epithelial cells (TECs) during kidney inflammation. CSF-1 mediates M?-dependent destruction in lupus-susceptible mice with nephritis and, paradoxically, M?-dependent renal repair in lupus-resistant mice after transient ischemia/reperfusion injury (I/R). We now report that I/R leads to defective renal repair, nonresolving inflammation, and, in turn, early-onset lupus nephritis in preclinical MRL/MpJ-Faslpr/Fas(lpr) mice (MRL-Fas(lpr) mice). Moreover, defective renal repair is not unique to MRL-Fas(lpr) mice, as flawed healing is a feature of other lupus-susceptible mice (Sle 123) and MRL mice without the Fas(lpr) mutation. Increasing CSF-1 hastens renal healing after I/R in lupus-resistant mice but hinders healing, exacerbates nonresolving inflammation, and triggers more severe early-onset lupus nephritis in MRL-Fas(lpr) mice. Probing further, the time-related balance of M1 "destroyer" M? shifts toward the M2 "healer" phenotype in lupus-resistant mice after I/R, but M1 M? continue to dominate in MRL-Fas(lpr) mice. Moreover, hypoxic TECs release mediators, including CSF-1, that are responsible for stimulating the expansion of M1 M? inherently poised to destroy the kidney in MRL-Fas(lpr) mice. In conclusion, I/R induces CSF-1 in injured TECs that expands aberrant M? (M1 phenotype), mediating defective renal repair and nonresolving inflammation, and thereby hastens the onset of lupus nephritis.     

Activated protein C attenuates systemic lupus erythematosus and lupus nephritis in MRL-Fas(lpr) mice

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease leading to inflammatory tissue damage in multiple organs (e.g., lupus nephritis). Current treatments including steroids, antimalarials, and immunosuppressive drugs have significant side effects. Activated protein C is a natural protein with anticoagulant and immunomodulatory effects, and its recombinant version has been approved by the U.S. Food and Drug Administration to treat severe sepsis. Given the similarities between overshooting immune activation in sepsis and autoimmunity, we hypothesized that recombinant activated protein C would also suppress SLE and lupus nephritis. To test this concept, autoimmune female MRL-Fas(lpr) mice were injected with either vehicle or recombinant human activated protein C from week 14-18 of age. Activated protein C treatment significantly suppressed lupus nephritis as evidenced by decrease in activity index, glomerular IgG and complement C3 deposits, macrophage counts, as well as intrarenal IL-12 expression. Further, activated protein C attenuated cutaneous lupus and lung disease as compared with vehicle-treated MRL-Fas(lpr) mice. In addition, parameters of systemic autoimmunity, such as plasma cytokine levels of IL-12p40, IL-6, and CCL2/MCP-1, and numbers of B cells and plasma cells in spleen were suppressed by activated protein C. The latter was associated with lower total plasma IgM and IgG levels as well as lower titers of anti-dsDNA IgG and rheumatoid factor. Together, recombinant activated protein C suppresses the abnormal systemic immune activation in SLE of MRL-Fas(lpr) mice, which prevents subsequent kidney, lung, and skin disease. These results implicate that recombinant activated protein C might be useful for the treatment of human SLE.     

Therapy for pneumonitis and sialadenitis by accumulation of CCR2-expressing CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript>regulatory T cells in MRL/lpr mice

Adoptive transfer of CD4⁺CD25⁺ regulatory T cells has been shown to have therapeutic effects in animal models of autoimmune diseases. Chemokines play an important role in the development of autoimmune diseases in animal models and humans. The present study was performed to investigate whether the progression of organ-specific autoimmune diseases could be reduced more markedly by accumulating chemokine receptor-expressing CD4⁺CD25⁺ regulatory T cells efficiently in target organs in MRL/MpJ-lpr/lpr (MRL/lpr) mice. CD4⁺CD25⁺Foxp3⁺ T cells (Treg cells) and CD4⁺CD25⁺Foxp3⁺ CCR2-transfected T cells (CCR2-Treg cells) were transferred via retro-orbital injection into 12-week-old MRL/lpr mice at the early stage of pneumonitis and sialadenitis, and the pathological changes were evaluated. Expression of monocyte chemoattractant protein-1 (MCP-1)/CCL2 was observed in the lung and submandibular gland of the mice and increased age-dependently. The level of CCR2 expression and MCP-1 chemotactic activity of CCR2-Treg cells were much higher than those of Treg cells. MRL/lpr mice to which CCR2-Treg cells had been transferred showed significantly reduced progression of pneumonitis and sialadenitis in comparison with MRL/lpr mice that had received Treg cells. This was due to more pronounced migration of CCR2-Treg cells and their localization for a longer time in MCP-1-expressing lung and submandibular gland, resulting in stronger suppressive activity. We prepared chemokine receptor-expressing Treg cells and demonstrated their ability to ameliorate disease progression by accumulating in target organs. This method may provide a new therapeutic approach for organ-specific autoimmune diseases in which the target antigens remain undefined.     

Leukocyte-endothelial cell interactions are enhanced in dermal postcapillary venules of MRL/fas(lpr) (lupus-prone) mice: roles of P- and E-selectin

MRL/fas(lpr) mice are affected by a systemic autoimmune disease that results in widespread leukocytic infiltration of the vasculature, including in the skin. The molecular pathways responsible for this leukocyte recruitment are poorly understood. Therefore, the aim of these experiments was to examine the mechanisms of leukocyte trafficking in the dermal microvasculature of MRL/fas(lpr) mice. Intravital microscopy was used to examine leukocyte rolling and adhesion in dermal postcapillary venules of MRL/fas(lpr) mice at 8, 12, and 16 wk of age. When compared with age-matched BALB/c and MRL(+/+) (nondiseased) mice, leukocyte rolling and adhesion in MRL/fas(lpr) mice were significantly enhanced at 12 wk of age, and remained elevated at 16 wk of age. At 8 and 12 wk, leukocyte rolling in all three strains was almost entirely inhibited by an anti-P-selectin mAb. In contrast, at 16 wk some (approximately 10%) leukocyte rolling persisted following P-selectin blockade. This residual rolling was predominantly inhibitable with an anti-E-selectin mAb; however, treatment with anti-E-selectin mAb alone had a minimal effect. P-selectin-deficient MRL/fas(lpr) mice also displayed leukocyte rolling that was significantly lower than in wild-type MRL/fas(lpr) mice. However, in these mice, leukocyte adhesion remained at the elevated levels observed in wild-type MRL/fas(lpr) mice. This adhesion was eliminated by chronic treatment with anti-E-selectin mAb. These findings indicate that leukocyte-endothelial cell interactions are enhanced in the dermal microvasculature of MRL/fas(lpr) mice above the age of 12 wk. Furthermore, the data suggest that the endothelial selectins share overlapping roles in mediating this enhanced leukocyte recruitment.     

IL-12 drives IFN-gamma-dependent autoimmune kidney disease in MRL-Fas(lpr) mice

IL-12 is secreted by kidney tubular epithelial cells in autoimmune MRL-Fas(lpr) mice before renal injury and increases with advancing disease. Because IL-12 is a potent inducer of IFN-gamma, the purpose of this study was to determine whether local provision of IL-12 elicits IFN-gamma-secreting T cells within the kidney, which, in turn, incites injury in MRL-Fas(lpr) mice. We used an ex vivo retroviral gene transfer strategy to construct IL-12-secreting MRL-Fas(lpr) tubular epithelial cells (IL-12 "carrier cells"), which were implanted under the kidney capsule of MRL-Fas(lpr) mice before renal disease for a sustained period (28 days). IL-12 "carrier cells" generated intrarenal and systemic IL-12. IL-12 fostered a marked, well-demarcated accumulation of CD4, CD8, and double negative (CD4-CD8- B220+) T cells adjacent to the implant site. We detected more IFN-gamma-producing T cells (CD4 > CD8 > CD4-CD8- B220+) at 28 days (73 +/- 14%) as compared with 7 days (20 +/- 8%) after implanting the IL-12 "carrier cells;" the majority of these cells were proliferating (60-70%). By comparison, an increase in systemic IL-12 resulted in a diffuse acceleration of pathology in the contralateral (unimplanted) kidney. IFN-gamma was required for IL-12-incited renal injury, because IL-12 "carrier cells" failed to elicit injury in MRL-Fas(lpr) kidneys genetically deficient in IFN-gamma receptors. Furthermore, IFN-gamma "carrier cells" elicited kidney injury in wild-type MRL-Fas(lpr) mice. Taken together, IL-12 elicits autoimmune injury by fostering the accumulation of IFN-gamma-secreting CD4, CD8, and CD4-CD8- B220+ T cells within the kidney, which, in turn, promote a cascade of events culminating in autoimmune kidney disease in MRL-Fas(lpr) mice.     

Characterization of reciprocal Lmb1-4 interval MRL-Faslpr and C57BL/6-Faslpr congenic mice reveals significant effects from Lmb3

Susceptibility to severe lupus in MRL-Fas(lpr) mice requires not only the lpr mutation but also other predisposing genes. Using (MRL-Fas(lpr) x B6-Fas(lpr))F2 (where B6 represents C57BL/6) intercrosses that utilize the highly susceptible MRL and poorly susceptible B6 backgrounds, we previously mapped CFA-enhanced systemic lupus-like autoimmunity to four loci, named Lmb1-4, on chromosomes 4, 5, 7, and 10. In the current study, we generated and analyzed reciprocal interval congenic mice for susceptibility to CFA-enhanced autoimmunity at all four Lmb loci. Although all loci had at least a slight effect on lymphoproliferation, only Lmb3 demonstrated a major effect on lymphoproliferation and anti-chromatin Ab levels. Further characterization of Lmb3, primarily by comparing MRL-Fas(lpr) with MRL.B6-Lmb3 Fas(lpr) congenic mice, revealed that it also played a significant role in spontaneous lupus, modifying lymphoproliferation, IgG and autoantibody levels, kidney disease, and survival. The less susceptible B6 Lmb3 locus was associated with a marked reduction in numbers of CD4(+) and double-negative (CD4(-)CD8(-)) T cells, particularly in lymph nodes, as well as reduced T cell proliferation and enhanced T cell apoptosis, both in vivo and in vitro. IFN-gamma-producing CD4(+) T cells were also reduced in MRL.B6-Lmb3 Fas(lpr) mice. Further mapping using subinterval congenic mice placed Lmb3 in the telomeric portion of chromosome 7. Thus, Lmb3, primarily through its effects on CD4(+) and double-negative T cells, appears to be a highly penetrant lupus-modifying locus. Identification of the underlying genetic alteration responsible for this quantitative trait locus should provide new insights into lupus-modifying genes.     

Membranous glomerulonephritis development with Th2-type immune deviations in MRL/lpr mice deficient for IL-27 receptor (WSX-1)

MRL/lpr mice develop spontaneous glomerulonephritis that is essentially identical with diffuse proliferative glomerulonephritis (World Health Organization class IV) in human lupus nephritis. Lupus nephritis is one of the most serious complications of systemic lupus erythematosus. Diffuse proliferative glomerulonephritis is associated with autoimmune responses dominated by Th1 cells producing high levels of IFN-gamma. The initial mounting of Th1 responses depends on the function of the WSX-1 gene, which encodes a subunit of the IL-27R with homology to IL-12R. In mice deficient for the WSX-1 gene, proper Th1 differentiation was impaired and abnormal Th2 skewing was observed during infection with some intracellular pathogens. Disruption of the WSX-1 gene dramatically changed the pathophysiology of glomerulonephritis developing in MRL/lpr mice. WSX-1-/- MRL/lpr mice developed disease resembling human membranous glomerulonephritis (World Health Organization class V) with a predominance of IgG1 in glomerular deposits, accompanied by increased IgG1 and IgE in the sera. T cells in WSX-1-/- MRL/lpr mice displayed significantly reduced IFN-gamma production along with elevated IL-4 expression. Loss of WSX-1 thus favors Th2-type autoimmune responses, suggesting that the Th1/Th2 balance may be a pivotal determinant of human lupus nephritis development.